Ribosomal RNA transcription regulates splicing through ribosomal protein RPL22

Ribosome biosynthesis is a cancer vulnerability targeted by inhibiting RNA polymerase I (Pol I) transcription. We developed specific Pol I inhibitors that activate a ribotoxic stress pathway to uncover drivers of sensitivity. Integrating multi-omics and drug response data from a large cancer cell panel, we found that RPL22 frameshift mutations confer Pol I inhibitor sensitivity. Mechanistically, RPL22 interacts directly with 28S rRNA and mRNA splice junctions, acting as a splicing regulator. RPL22 deficiency, intensified by 28S rRNA sequestration, promotes splicing of its paralog RPL22L1 and the p53 negative regulator MDM4. Both chemical and genetic inhibition of rRNA synthesis broadly remodel mRNA splicing controlling hundreds of targets. Notably, RPL22-dependent alternative splicing is reversed by Pol I inhibition, revealing a non-canonical ribotoxic stress-initiated tumor suppressive pathway. This study uncovers a robust mechanism linking rRNA synthesis activity to splicing, coordinated by the ribosomal protein RPL22.